Methods for enhancing the production of interferon in cell culture

ABSTRACT

Methods for enhancing the production of interferons in animal cell culture are described. These methods rely on the manipulation of the cellular levels of certain inducers of interferon production, in particular cellular levels of double-stranded-RNA-dependent kinase (dsRNA-PKR, or PKR). In cell cultures that overproduce PKR, interferon synthesis is induced to high levels, and significant amounts of interferon can be recovered without conventional induction of interferon by virus.

This application is a continuation of U.S. application Ser. No.08/701,136, filed Aug. 21, 1996, which claims the benefit of U.S.Provisional Application No. 60/002,621, filed Aug. 22, 1995.

TECHNICAL FIELD

The present invention relates to methods for enhancing the production ofinterferon in cell culture.

BACKGROUND

Interferons (IFNs) can be classified into two major groups based ontheir primary sequence. Type I interferons, IFN-α and IFN-β, are encodedby a super family of intronless genes consisting of the IFN-α genefamily and a single IFN-β gene. Type II interferon, or IFN-γ, consistsof only a single type and is restricted to lymphocytes (T-cells andnatural killer cells). Type I interferons mediate diverse biologicalprocesses including induction of antiviral activities, regulation ofcellular growth and differentiation, and modulation of immune functions(Sen, G. C. & Lengyel, P. (1992) J. Biol. Chem. 267, 5017-5020; Pestka,S. & Langer, J. A. (1987) Ann. Rev. Biochem. 56, 727-777). The inducedexpression of Type I IFNs, which include the IFN-α and IFN-β genefamilies, is detected typically following viral infections. Many studieshave identified promoter elements and transcription factors involved inregulating the expression of Type I IFNs (Du, W., Thanos, D. & Maniatis,T. (1993) Cell 74, 887-898; Matsuyama, T., Kimura, T., Kitagawa, M.,Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R.,Kishihara, K., Wakeham, A., Potter, J., Furlonger, C. L., Narendran, A.,Suzuki, H., Ohashi, P. S., Paige, C. J., Taniguchi, T. & Mak, T. W.(1993) Cell 75, 83-97; Tanaka, N. & Taniguchi, T. (1992) Adv. Immunol.52, 263-81). However, it remains unclear what are the biochemical cuesthat signify viral infections to the cell and the signaling mechanismsinvolved (for a recent review of the interferon system, see Jaramillo etal. Cancer Investigation 1995 13:327-337).

IFNs belong to a class of negative growth factors having the ability toinhibit growth of a wide variety of cells with both normal andtransformed phenotypes. IFN therapy has been shown to be beneficial inthe treatment of human malignancies such as Kaposi's sarcoma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma and hairy cell leukemia aswell as the treatment of infectious diseases such as papilloma virus(genital warts) and hepatitis B and C (reviewed by Gutterman Proc. NatlAcad Sci. 91:1198-1205 1994). Recently genetically engineered,bacterially produced IFN-β was approved for treatment of multiplesclerosis, a relatively common neurological disease affecting at least250,000 patients in the United States alone.

Currently, IFNs for therapeutic use are produced from two major sources,natural IFNs from human leukocytes or leukocyte cell lines andrecombinant IFNs produced in bacterial cells. Natural IFNs areconsidered to be superior as they consist of the entire complement ofIFNs and have the proper structure, but they are expensive andtime-consuming to produce. Bacterially produced recombinant IFN ischeaper and more efficient to make, but studies have shown much higherrates of rejection for the bacterially produced protein, particularlyafter long term usage. For instance, previous medical studies have shownthat the incidence of rejection as reflected by antibody formation canbe as high as 20 to 38% for bacterially-produced IFN compared with only1.2% for natural IFN-α (Antonelli et al. J. Inf. Disease 163:882-8851991; Quesada et al. J. Clin. Oncology 3:1522-1528 1985). Thus, a methodfor enhancing the production of natural IFN to make it less expensive toproduce would be advantageous.

IFNs elicit their biological activities by binding to their cognatereceptors followed by signal transduction leading to induction ofIFN-stimulated genes (ISGs). Some ISGs have been characterized and theirbiological activities examined. The best studied examples of ISGsinclude a double-stranded-RNA-dependent kinase (dsRNA-PKR, or just PKR,formerly known as p68 kinase), 2'-5'-linked oligoadenylate (2-5A)synthetase, and Mx proteins (Taylor JL, Grossberg SE. Virus Research1990 15:1-26.; Williams BRG. Eur. J. Biochem. 1991 200:1-11.).

PKR (short for protein kinase, RNA-dependent) is the only identifieddsRNA-binding protein known to possess a kinase activity. PKR is aserine/threonine kinase whose enzymatic activation requires dsRNAbinding and consequent autophosphorylation (Galabru, J. & Hovanessian,A. (1987) J. Biol. Chem. 262, 15538-15544; Meurs, E., Chong, K.,Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R. G. &Hovanessian, A. G. (1990) Cell 62, 379-390). PKR has also been referredto in the literature as dsRNA-activated protein kinase, P1/e1F2 kinase,DAI or dsI (for dsRNA-activated inhibitor), and p68 (human) or p65(murine) kinase. Analogous enzymes have been described in rabbitreticulocytes, different murine tissues, and human peripheral bloodmononuclear cells (Farrel et al. (1977) Cell 11, 187-200; Levin et al.(1978) Proc. Natl Acad. Sci. USA 75, 1121-1125; Hovanessian (1980)Biochimie 62, 775-778; Krust et al. (1982) Virology 120, 240-246;Buffet-Janvresse et al. (1986) J. Interferon Res. 6, 85-96). The bestcharacterized in vivo substrate of PKR is the alpha subunit ofeukaryotic initiation factor-2 (eIF-2a) which, once phosphorylated,leads ultimately to inhibition of cellular and viral protein synthesis(Hershey, J. W. B. (1991) Ann. Rev. Biochem. 60, 717-755). Thisparticular function of PKR has been suggested as one of the mechanismsresponsible for mediating the antiviral and antiproliferative activitiesof IFN-α and IFN-β. An additional biological function for PKR is itsputative role as a signal transducer. Kumar et al. demonstrated that PKRcan phosphorylate IκBα, resulting in the release and activation ofnuclear factor κB (NF-κB) (Kumar, A., Haque, J., Lacoste, J., Hiscott,J. & Williams, B. R. G. (1994) Proc. Natl. Acad. Sci. USA 91,6288-6292). Given the well-characterized NF-κB site in the IFN-βpromoter, this may represent a mechanism through which PKR mediatesdsRNA activation of IFN-β transcription (Visvanathan, K. V. &Goodbourne, S. (1989) EMBO J. 8, 1129-1138).

The present inventor have surprisingly discovered that manipulating theexpression of certain ISGs can have beneficial uses in interferonproduction. They have discovered that over-expression of the PKR proteininduces overproduction of the IFN-α and IFN-β interferons, which isuseful for the enhanced production of interferon in animal cell culture.

Relevant Literature

Currently there are two major approaches to large-scale production ofinterferons: recombinant IFN produced in bacterial or mammalian cells ornatural IFNs from human leukocyte cells following stimulation withviruses or other IFN inducers. U.S. Pat. No. 5,376,567 and U.S. Pat. No.4,966,843 describe the production in Chinese hamster ovary cells of arecombinant human interferon; U.S. Pat. No. 5,196,323 describes theproduction of recombinant human IFN-α in E. coli cells. A number ofpatents describe the production of interferon from human leukocyte cellsusing a variety of protocols; for example, U.S. Pat. No. 4,745,053describes a process for producing interferon from whole human bloodusing a viral inducer, U.S. Pat. No. 4,680,261 describes a process forinducing production of interferon in mammalian cell culture using anascorbic acid derivative or an inorganic vanadium compound, and U.S.Pat. No. 4,548,900 describes a process for the induction of interferonusing a polyhydric alcohol in a priming stage. The major disadvantage ofthe current methods of interferon production is that typically virus isused as the IFN inducer because other inducers do not produce highenough levels of interferon for most commercial purposes. The virus mustthen be removed from the interferon before use, which adds time and costto the production method. In addition, use of virus as an inducerultimately results in the death of the interferon-producing cells, sothat no recycling and re-use of the cells is possible.

The present invention overcomes these problems by providing ainterferon-production system that does not require the use of a viralinducer in order to achieve high levels of interferon production.Although viral inducers can be used with the systems of the invention,other inducers that do require removal prior to use of the IFNs arestill capable of producing IFNs at commercially acceptable levels.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a method for enhancing theproduction of interferon in animal cell culture without requiring theaddition of virus as an inducer.

This object is generally accomplished by providing animal cell culturesin which the expression of the interferon genes is substantiallyincreased from the normal level of expression. This may be effected bymanipulating the level of expression of factors that function in vivo toregulate the interferon level, including interferon transcriptionalregulators (for example, IRF1), interferon receptors, and interferonstimulated gene products (for example PKR and 2-5A synthetase).

Thus, increased production of INF and other objects of the invention aswill hereinafter become apparent are accomplished by carrying outinterferon production in animal cell cultures in which the level ofinterferon-regulating protein activity, particularly fordouble-stranded-RNA-dependent kinase (PKR) and 2'-5' oligoadenylatesynthetase (2-5A synthetase), is significantly increased from normallevels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Diagrammatic drawings of pCMV-PKR and pMT-PKR. 1(A) Diagram ofthe pCMV-PKR showing the human PKR cDNA in the sense orientation drivenby the promoter sequence from the immediate early gene of humancytomegalovirus. 1(B) Diagram of the pMT-PKR showing the human PKR cDNAin the sense orientation driven by the cadmium inducible metallothioneinII promoter.

FIG. 2. Interferon induction in U937-PKR+ cells and control U937-neocells. U937-PKR+ cells and U937-neo cells (0.5×10⁶ /ml) were cultured inRPMI 1640 medium, supplemented with 5% fetal bovine serum. The cellswere pretreated ("primed") with or without 10 nM PMA for 20 hrs followedby induction with 10 μg/ml poly r(I):poly r(C) or with 0.1 TCID₅₀ / cellEMCV for 20 hrs. Twenty-four hours after induction, culture supernatantswere collected and assayed for IFN-α. The open bars indicate U937-neocells, the hatched bars indicate U937-PKR+ cells. The experimentalgroups indicate different treatments: 1- no treatment; 2- poly r(I):polyr(C) induction alone; 3- EMCV induction alone; 4- PMA priming alone; 5-PMA priming and poly r(I):poly r(C) induction; 6- PMA priming and EMCVinduction.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention arose in part from the discovery by the inventorthat the level of interferon production in cell culture can be regulatedby control of the expression or activity of certain proteins thatnormally regulate interferon expression in vivo. These factors includeinterferon-specific transcriptional regulators (particularly IRF1),interferon receptors, and the gene products of certain interferonsimulated genes (also called interferon-mediated antiviral responses),particularly PKR. Enhancing the expression or activity of any of thesefactors will result in a higher than normal level of expression ofinterferon genes. One result of this higher than normal expression ofinterferon genes is that interferon production can be achieved withgreater efficiency and lower cost. The remainder of this specificationwill be exemplified by reference to PKR, but it is to be understood thatother interferon enhancing factors as described herein can be used inplace of PKR.

By increasing the level of PKR protein (and therefore PKR activity) inan animal cell, interferon production can be increased. Animal cellcultures which express a higher constitutive level of PKR or in whichPKR expression can be induced to higher levels are therefore useful forthe production of interferons. Problems typically associated withproduction of interferons in cell culture, for example low yield andcontamination with the virus used to induce interferon expression, areeliminated in the method of the present invention. Thus, a high yield ofinterferon protein can be achieved without the need for induction withvirus (although virus can be used for even higher INF production).

The method relies on the use of PKR-overproducing cells as the source ofthe interferon, but any known or later-discovered method of interferonproduction may be used. No particular method of production of interferonis required except that typically a non-viral interferon inducer isused. In particular, the method comprises (a) culturing an animal cellculture capable of overproduction of PKR or an analog or homolog of PKRunder conditions sufficient to overproduce PKR, and (b) treating thecell culture as appropriate to induce the expression of the interferongenes, particularly the IFN-β and IFN-α. These steps are generallyfollowed by purifying the interferon produced. In some cases, theoverproduction is not of PKR itself, but of an analog of PKR, by whichis meant a non-natural protein kinase that can mediate dsRNA activationof interferon transcription (usually obtained by genetic manipulation ofthe gene encoding a particular PKR from a selected animal cell line).The cell culture used to produce interferon can overproduce PKR from anyanimal cell line used to produce the interferon, such as the PKRnormally found in rabbit reticulocytes, various mouse tissues, or humanperipheral blood mononuclear cells. Usually, the natural PKR for aparticular cell line (as opposed to an exogenous PKR) is used foroverexpression. Preferably murine p65 kinase and most preferably humanp68 kinase is overproduced, in a corresponding murine or human cellculture, respectively.

Animal cell cultures capable of overproduction of a PKR gene may beisolated by any number of methods, including many that are well known inthe art. Such methods include selection for cells expressing higher PKRlevels, transfection with a vector containing a PKR gene (or cDNA) undercontrol of a constitutive promoter (for example, a CMV, RSV or SV40promoter), or transfection with a vector containing a PKR gene or cDNAunder the control of an inducible promoter (for example, a heat shockpromoter, a metallothionein promoter or a glucocorticoid promoter).Transfection is carried out as described previously and transfectantsare selected for overproduction of PKR. By overproduction of PKR ismeant higher than normal levels of PKR activity. Normal refers to undernormal conditions for the cell line being used (see, for example,suggested culture conditions as supplied in catalogues for commercialcells, especially the ATCC Catalogue of Strains, available from theAmerican Type Culture Collection, Rockville, Md., USA, or in scientificpublications describing the cell line being used, if it is notcommercially available), and in various preferred embodiments refers toproduction of INF under otherwise identical conditions in the parentalcell line from which a production cell line is selected or produced bygenetic engineering. Higher than normal level preferably means at least150%, more preferably at least 200 or 300%, most preferably at least500%, of normal PKR level. The PKR-overproducing cell culture may beconstitutive for PKR overproduction or inducible for PKR overproduction,depending on the particular method used to isolate or prepare theculture. Preferably the PKR-overproducing cell culture will be induciblefor PKR overproduction in order to regulate the level of PKR availablefor IFN induction. It will be apparent that if the PKR-overproducingcell culture is inducible for overproduction, PKR activity foroverproduction will be assayed under inducing conditions. PKR activitycan be determined by any of the methods known in the art or described inthe following examples.

Any of a number of known cell cultures are useful as a parental strainfor making a PKR-overproducing cell culture. Any cells normally capableof producing interferon are suitable as the parental strain,particularly cells derived from fibroblasts or immune cells, including Bcells and monocytes. Particularly suitable cell cultures arepro-monocytic U937 cells, Namalwa (lymphoblastoid B) cells and MRC-5(human fibroblast) cells. Also suitable are WI-38 cells, Flow 1000cells, Flow 4000 cells, FS4 and FS-7 cells, MG-63 cells, CCRF-SB cellsand CCRF-CEM cells.

Production of interferon in the PKR-overproducing cell culture isaccomplished by methods that are well known in the art. Generally, theinterferon producing cells are cultured in any suitable medium, treatedwith an inducer to induce expression of the interferons (and with aninducer of the PKR gene, in PKR is provided under the control of aninducible promotor), and incubated following induction of interferonproduction. The interferon is then isolated. The cells may be primedprior to induction by addition of a priming agent. Interferon inducersare many and well -known in the art and almost any known interferoninducer can be used in the present invention. Typical inducers includepoly(I):poly(C), Sendai virus, Newcastle disease virus, concanavaline A,chlamydia, rickettsia, mitogen, and lipopolysaccharides. Preferably, theinducer will be a non-viral inducer. More preferably, the inducer willbe poly(I):poly(C) or poly r(I):poly r(C). Non-viral inducers arepreferred because the cells do not suffer the deleterious consequencesthat exposure to the virus provides and may therefore be recycled forre-use resulting in a lower cost of production. Typical priming agentsinclude phorbol myristate acetate, calcium ionophores and interferons.

IFNs are purified by standard techniques that are well known in the art.These include antibody-affinity column chromatography, ion exchangechromatography, protein precipitation and centrifugation (U.S. Pat. No.5,391,713); chromatography using CM-agarose, con A-agarose, andphenyl-agarose (U.S. Pat. No. 4,658,017); three step chromatography withglass sorbent column (U.S. Pat. No. 4,732,683); immunoaffinitychromatography, reverse phase HPLC, cation exchange column, and gelfiltration (U.S. Pat. No. 4,765,903); guanidine HCl as solvent and HICcolumn chromatography (U.S. Pat. No. 4,845,032).

The levels of IFN-α in the preparations can be determined by titratingfor antiviral activity on L929 (murine fibroblast) cells againstNational Institute of Allergy and Infectious Diseases reference standardG-023-901-527 (Lau et al. J. Clin. Invest. 82:1415-1421 1988). In thistechnique, 5×10⁴ L929 cells are seeded into each well of 96-wellmicrotiter plates and then incubated with two-fold serial dilutions ofculture supernatants for 16 hr. The cells are subsequently challengedwith EMCV or Sendai virus (0.1 or 0.01 TCID per cell). Virus-inducedcytopathic effects were assessed by microscopic examination and bystaining cells with 0.1% crystal violet in 5% ethanol solutions. The IFNtiter is defined as the reciprocal of the highest dilution of culturesupernatants capable of protecting 50% of the cells from viral-inducedcytopathic effects.

Specific examples of the steps described above are set forth in thefollowing examples. However, it will be apparent to one of ordinaryskill in the art that many modifications are possible and that theexamples are provided for purposes of illustration only and are notlimiting of the invention unless so specified.

EXAMPLES Example 1: Preparation of Constitutive PKR Overexpressing CellCulture

Plasmid pCMV-PKR was prepared from pRC/CMV by inserting the PKR cDNAfrom pBS-8.6R into the HindIII site of the pRC/CMV vector so thatexpression of the PKR coding sequence is under control of the CMVpromoter. The pRC/CMV plasmid (Invitrogen) is commonly used foreukaryotic expression. The vector offers the following features suitablefor PKR transcription: i) promoter sequences from the immediate earlygene of the human CMV (cytomegalovirus) for high level transcription;ii) polyadenylation signal and transcription termination sequences frombovine growth hormone (BGH) gene to enhance RNA stability; iii) SV40origin for episomal replication and simple vector rescue; iv) T7 and Sp6RNA promoters flanking the multiple cloning site for in vitrotranscription of sense and antisense RNA; and v) the ampicillinresistance gene (AMP) and ColE1 origin for selection and maintenance inE. coli. The vector also contains a G418 resistance marker (NEO) toallow for selection and identification of the plasmids after transfer toeukaryotic cells. The structure of pCMV-PKR is shown in FIG. 1A.

Stable transfectants were obtained by electroporation of 5×10⁶exponentially growing U937 cells with 10 mg of each plasmid, inserum-free RPMI-1640 containing DEAE-dextran (50 mg/mL), with a GenePulser apparatus (BioRad) set at 500 μF, 250V. Bulk populations ofstable transfectants were obtained by selection with 400 μg/mL geneticin(GIBCO-BRL) for 3 weeks. Clonal lines were subsequently obtained bylimiting dilution cloning. Cell lines were cultured in RPMI-1640containing 10% fetal calf serum (complete media) and geneticin. Onerepresentative transfectant cell line was selected and designated"U937-PKR+". The level of PKR produced in U937-PKR+ was analyzed andfound to be increased approximately five-fold over normal (parental)levels. As a control, U937 cells were transfected with pRC/CMV and arepresentative transfectant cell line was isolated and designated"U937-neo". The transfectant cell lines were tested for IFN productionin the presence or absence of priming agents and/or inducers.

U937-PKR+ cells and U937-neo cells were cultured in RPMI 1640 medium,supplemented with fetal bovine serum. The cells were pretreated with orwithout phorbol myristate acetate (PMA) (10nM) for 20 hr and with orwithout subsequent stimulation by poly r(I):r(C), 10 ug/ml, for 20 hr.Twenty-four hr after the stimulation with poly r(I):r(C), culturesupernatants were collected for IFN assays.

As shown in FIG. 2, neither transfectant cell line produced IFNspontaneously in the absence of priming agents or inducers. In responseto poly r(I):poly r(C), control U937-neo cells showed minimal synthesisof IFN-α either with or without PMA priming (open bar 2 and 5). Incontrast, U937-PKR+ cells showed increased IFN production in response topoly r(I):poly r(C) induction, with or without PMA priming, reaching alevel of 4000 U/ml when treated with both poly r(I):poly r(C) and PMA(hatched bars 2 and 5). In the absence of PMA priming, EMCV induced highlevels of IFN in both transfectant cell lines (more than 1000 U/ml),with slightly higher levels in the U937-PKR+cells (Bar 3). In theabsence of EMCV or poly r(I):poly r(C) inducer, U937-PKR+ cells producedhigher levels of IFNs than did U937-neo cells in response to PMA primingalone (Bar 4). With PMA priming and EMCV induction, both transfectantcell lines produced more than 4000 U/ml IFN (Bar 6). Taken together,these results indicated that following priming with PMA, the U937-PKR+cells are as efficient in IFN production when poly r(I):poly r(C) isused as inducer as are control cells when virus is used as inducer.Therefore the use of live virus for IFN production can be eliminatedwhen PKR-overproducing cells are used.

Following induction by poly r(I):poly r(C), the U937-PKR+ cells wereexamined for viability using a trypan blue exclusion assay. We foundthat more than 95% of the cells were viable. In contrast, the use ofEMCV as an inducer resulted in the death of all of the cells. Thus, theU937-PKR+ cells can be recycled for continuous production of IFN.

Example 2: Preparation of Inducible PKR Overexpressing Cell Culture

Plasmid pMT-PKR was made by replacing the CMV promoter upstream from thePKR cloning site in pCMV-PKR with a cadmium-inducible metallothionein IIpromoter (Hewison et al. J. Immunol. 153:5709-5719 1994). The structureof pMT-PKR is shown in FIG. 1B. Stable transfectants of pMT-PKR intoU937 cells were isolated as described in Example 1 for pCMV-PKRtransfectants. The MTII promoter is inducible by zinc or cadmium ions.PKR activity levels in the transfectants were measured after inductionof the transfected PKR gene by addition of 20 μM cadmium chloride.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the describedinvention.

What is claimed is:
 1. A method for producing α- or β-interferon in ahuman cell culture, in the absence of viral induction, comprising:(a)culturing a human cell line capable of producing interferon andtransfected with a vector containing DNA encodingduble-stranded-RNA-dependent kinase PKR under the control of a promoter,under culture conditions in which PKR is overproduced in the transfectedcells, as evidenced by levels of PKR in said transfected cell line whichare higher than those obtained in said human cell line which is nottransfected with said vector, when grown under the same cultureconditions, (b) treating said cultured, PKR-overproducing human cellline with double-stranded RNA (dsRNA), in the absence of viralinduction, and (c) collecting α- or β-interferon produced by thecultured, treated cell line.
 2. The method of claim 1, wherein saidculturing includes priming the cells with phorbal myristate acetate(PMA).
 3. The method of claim 1, wherein said collecting includesisolating α-interferon produced by the cultured, treated cells.
 4. Themethod of claim 1, wherein said collecting includes isolatingβ-interferon produced by the cultured, treated cells.